C-di-GMP信号通路和RgpAc基因调节变形链球菌致龋特性的初步研究
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摘要
龋病是人类最常见的细菌感染性疾病之一,发病率高,涉及范围广。变形链球菌(Streptococcus mutans,S. mutans)作为龋病主要的致病菌,拥有复杂的毒力因子,参与了其在牙面的黏附、聚集、菌斑生物膜的形成、产酸耐酸等作用。
比较基因组学研究发现一类含有GGDEF/EAL结构域的蛋白在各类细菌中普遍存在,进一步研究发现其功能主要是参与3'-5'环二鸟苷酸(bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP)的合成与分解,该结构域能够催化两个GTPs合成c-di-GMP。大部分的GGDEF结构域在临近N端的活性位点处含有一个Ⅰ位点,该位点可以与非竞争性抑制产物c-di-GMP结合,这种别构抑制作用能够限制c-di-GMP的浓度,阻止GTP的过度消耗。磷酸二酯酶(phosphodiesterases, PDEs)上的EAL结构域具有水解c-di-GMP的作用,其水解产物是线性的5'-磷酸鸟苷酸-(3’-5')-鸟苷(5'-phosphoguany1y1-(3'-5')-guanosine,5'-pGpG)分子,这些小分子物质不具备生物活性,在细胞内很快被其它的磷酸二酯酶降解。通常情况下,GGDEF和EAL结构域存在于同一个蛋白的C端,而负责信号感知及传导的结构域则位于蛋白的N端,可以感知一系列信号,例如PAS和GAF结构域,涉及到细胞内小分子的结合或者蛋白与蛋白之间的相互作用。除此之外,还有一小部分GGDEF和EAL结构域蛋白的N端整合在细胞膜上,负责感知信号,但是目前还不清楚这些结构域能够感知什么样的信号。
近年来的研究已经证实,c-di-GMP是原核细胞信号转导中一类新的第二信使,是一种小分子复合物,具有热稳定性,能够激活纤维素合成酶,参与多种复杂的生理活动的调控。它的作用涉及细菌细胞的分化、生物膜的形成、细胞间的通讯、毒力因子的表达以及与宿主细胞间相互作用等多个方面,是细菌生存和代谢过程中的关键性调节因子之一。
研究发现变形链球菌中存在c-di-GMP信号通路,外源性c-di-GMP的加入和内源性c-di-GMP的丧失均会降低变形链球菌的致龋特性(表现为生物膜形成量减少),但其机制尚不清楚。对这些问题进一步的研究将有助于加深对变形链球菌中c-di-GMP信号途径的作用方式的认识,也为探索新的防龋方法提供了依据。本课题组在前期研究的基础上,构建了变形链球菌内gcp基因敲除菌株,以便更全面、深入的探讨变形链球菌内c-di-GMP的作用机制和信号通路,同时与S. mutans UA159野生菌株进行基因芯片比较,筛选和生物膜有关的基因进行信号通路分析。
本论文包括以下三部分内容：
第一章：变形链球菌gcp基因突变株的构建
本实验根据NCBI提供的S. mutans UA159全基因组序列,采用Primer Premier5.0软件(Premier公司)设计gcp基因的上游引物和下游引物,扩增gcp基因的上游片段和下游片段。采用双酶切及同源重组的方式构建了含有壮观霉素抗性(spe)的载体pFW5-gcp-UP-DOWN。在下一步实验中,采用同源重组的方式构建变形链球菌gcp基因突变菌株。
用限制性内切酶Nhe Ⅰ酶(NEB公司)对载体pFW5-gcp-UP-DOWN进行酶切,将环状的质粒变为线性化的片段,胶回收酶切后的线性化片段10ug备用。取1ml变形链球菌感受态细胞菌液,加入30μl线性化的重组载体pFW5-gcp-UP-DOWN,采用同源重组的方式构建变形链球菌gcp基因突变菌株,命名为gcp-KO。
通过对转化菌的形态学观察发现,转化菌形态与S. mutans UA159菌基本一致,但是细菌排列更为稀疏,这可能是由于基因缺陷后引起的生长差异所致。对重组载体进行双酶切鉴定结果显示：重组载体pFW5-gcp-UP经过Sal Ⅰ和Bgl Ⅱ双酶切后出现2条带,与质粒pFW5-gcp-UP的大小基本相同。重组载体pFW5-gcp-UP-DOWN经过Nco Ⅰ和Nde Ⅰ双酶切后出现2条带,与质粒pFW5-gcp-UP及gcp-DOWN的大小基本相同。对野生菌与转化菌的gcp基因上下游片段、spe基因进行PCR扩增及电泳鉴定结果显示：野生菌株不能扩增spe基因,而转化菌株能够扩增上、下游基因及spe基因,表明spe基因成功转入菌株基因组,测序结果也显示gcp基因被spe基因所同源替代。
第二章：变形链球菌gcp基因突变菌株表达谱基因芯片研究及验证
提取变形链球菌gcp-KO及野生菌株的总RNA,参照大连宝生物工程公司1st Strand cDNA Synthesis Kit试剂盒说明书分别合成cDNA。对照组(野生菌株)加入Cy5-CTP*(10mmol/L),实验组(突变菌株)加入Cy3-CTP*(10mmol/L)进行标记,并采用3S柱PCR产物纯化试剂盒纯化,得到杂交混合液。吸取适量杂交混合液,使样品液均匀的覆盖在玻片的表面,检查气泡后将基因芯片字符面朝上放入芯片夹中,将芯片夹置入基因芯片扫描仪NimbleGen MS200中,采用MS200Data Collection软件,由仪器自动平衡红绿荧光的激发光能量及增益值进行扫描,获得双色叠加的杂交结果图,保存该结果。
本实验为了确定差异基因表达谱,采用国内外基因芯片实验常用标准Ratio'=2做为差异基因的初步筛选标准。上调基因筛选的转录差异要在2倍以上,即FC≥2。同样下调基因筛选的转录差异也是2倍以上,即FC≤0.5。转录差异在0.5≤FC≤2之间的基因,被认为没有差别,不筛选进入下一步研究,最终有411个基因符合条件。分别比较两组数据中Cy5荧光信号强度和Cy3荧光信号强度,对于同一个探针,如果Cy5信号强度是Cy3的2倍或2倍以上,认为该探针所代表的基因表达降低；如果Cy3的信号强度是Cy5的2倍或2倍以上,说明该探针代表的基因表达升高。初步筛选411个差异基因,表达上调的基因198个,表达下调的基因213个。再结合GO分析、COG分析及KEGG分析的结果,最终确定8个基因进入本课题的Real-time PCR验证研究,其中上调基因和下调基因各4个。表达差异的基因按照功能进行归类,查阅GenBank数据库及相关文献对其结果进行深入的分析及讨论。结合前期实验的结果,选择RgpAc基因作为后续研究的目的基因。
第三章：RgpAc基因突变株的构建及功能研究
本章主要包括两方面的内容：
1.RgpAc基因突变株的构建
本实验根据NCBI提供的S. mutans UA159全基因组序列,采用Primer Premier5.0软件设计RgpAc基因的上游引物和下游引物,扩增RgpAc基因的上游片段和下游片段。采用双酶切及同源重组的方式构建了含有壮观霉素抗性(spe)的载体pFW5-RgpAc-UP-DOWN。将载体加入变形链球菌感受态细胞中,采用同源重组的方式构建变形链球菌RgpAc基因突变菌株,命名为RgpAc-KO。通过对转化菌的形态学观察发现,转化菌形态与S. mutans UA159菌基本一致,但是细菌排列更为稀疏,这可能是由于基因缺陷后引起的生长差异所致。实验还对野生菌与转化菌的RgpAc基因及其上下游、spe基因进行了PCR扩增与电泳鉴定。结果显示,野生菌株含有RgpAc基因,不含有spe基因。而转化菌株能够扩增载体的上、下游基因及spe基因,不能扩增RgpAc基因,表明转化菌株有RgpAc基因缺陷。测序结果也显示RgpAc基因被spe基因所同源替代。
2.变形链球菌RgpAc基因功能的研究
采用SPSS13.0统计学软件进行统计学处理,对比了野生菌株和突变菌株在24h、48h形成的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖的含量,以及野生菌株和突变菌株加入不同浓度的c-di-GMP之后,形成的生物膜含量。采用析因设计的方差分析的方法,各组计量资料数据采用均数±标准差的方式列出,检验水准a=0.05。
通过改良蒽酮比色法比较突变菌株和野生菌株胞外多糖的合成能力。实验分为两组,分别为野生菌株和突变菌株,24h的样本量为8例,48h的样本量为5例。野生菌株水溶性葡聚糖和胞外多糖生成量高于突变菌株,差异有统计学意义(F=328.868, P<0.001; F=80.897, P<0.001),突变菌株水不溶性葡聚糖生成量高于野生菌株,差异有统计学意义(F=15.718,P<0.001)。
同一菌株不同的时间段相比,野生菌株24h和48h产生的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖差异均有统计学意义(F=36.022,P<0.001；F=15.274, P<0.001; F=-18.241, P<0.001),24h处产生的水溶性葡聚糖高于48h处,水不溶性葡聚糖低于48h处,胞外多糖高于48h处。突变菌株24h和48h产生的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖差异均有统计学意义(F=-7.537, P<0.001; F=18.762, P<0.001; F=-8.587, P<0.001),24h产生的水溶性葡聚糖高于48h处,水不溶性葡聚糖低于48h处,胞外多糖低于48h处。
在比较生物膜形成量的实验中,S.mutans UA159与RgpAc-KO加入200μM和400μM的c-di-GMP后,设计了6组,每组有5例,采用析因分析结果发现,两组菌株之间的差异有统计学意义(F=29.411,P<0.001),突变菌株高于野生菌株；三个c-di-GMP浓度间的差异有统计学意义(F=3.945, P=0.033),400μM组最高,空白对照组最低；在不加c-di-GMP干扰的时候,野生菌株和突变菌株之间的差异有统计学意义(t=8.196,P<0.001),野生菌株更高；在加入200μM、400μM的c-di-GMP干扰后,野生菌株和突变菌株之间的差异有统计学意义(t=-4.467, P=0.002;t=-8.038, P<0.001),突变菌株均高于野生菌株；野生菌株加入不同浓度的c-di-GMP干扰后,产生的差异有统计学意义(F=24.766,P<0.001),不加c-di-GMP组最高,加入400μM的c-di-GMP组最低；突变菌株加入不同浓度c-di-GMP干扰后,产生的差异有统计学意义(F=48.322,P<0.001),不加c-di-GMP组最低,加400μM的c-di-GMP组最高;交互效应有统计学意义(F=47.529,P<0.001)。实验结果表明变形链球菌RgpAc基因敲除后,细菌形成生物膜的能力被抑制。在变形链球菌中,各种水溶性和水不溶性的多糖是生物膜形成的重要基质之一。当变形链球菌的RgpAc基因敲除后,合成水溶性葡聚糖和胞外多糖的能力均下降,导致生物膜形成的基质缺乏,最终抑制了生物膜的形成。突变菌株加入不同浓度的c-di-GMP之后,生物膜形成能力增加,推测原因是外源性的c-di-GMP增加后促进了其与下游信号通路受体的结合,最终使生物膜的形成量增加。
为了比较两种细菌在牙齿表面形成的生物膜结构的差异,实验模拟体内情况在釉质表面形成人工生物膜,并进行了扫描电镜观察。结果显示,RgpAc-KO与野生菌株培养48h后,产生的生物膜形态不相同。低倍镜下可见S. mutansUA159组产生的生物膜较厚,排列较致密,RgpAc-KO组的生物膜结构较松散,突变菌株分别加入200μM和400μM的c-di-GMP之后,细胞外基质增加。高倍视野下可见S. mutans UA159组细菌之间有较多的细胞外基质,生物膜上的细菌排列规律,形成漩涡状结构；RgpAc-KO组细菌之间的细胞外基质较少,视野内可见少量的细菌。突变菌株分别加入200μM和400μM的c-di-GMP之后,细菌生物膜的结构变致密,细胞数目增多。提示RgpAc基因突变后降低了水溶性葡聚糖的合成,而水溶性葡聚糖既可作为细菌胞外能源物质及代谢底物,又可与细菌表面的Gbp结合,能够促进黏附和聚集。因此在电镜下观察突变菌株的生物膜结构较松散,细胞外基质较少,视野内细菌数量少。加入外源性c-di-GMP后增加了下游信号通路的活性,最终使生物膜的形成增加。
综上,本实验通过同源重组的方法构建了变形链球菌gcp基因突变菌株,与野生菌株进行了基因芯片比较,表达差异在2倍以上的基因有411个,包括198个表达上调的基因和213个表达下调的基因。通过查阅资料,最终挑选8个最有可能与变形链球菌生物膜形成及信号通路有关的基因进行验证,发现RgpAc基因表达抑制的程度最大,因此课题组选择该基因作为下一步研究的目的基因,构建了变形链球菌RgpAc基因突变菌株。利用比色、电镜扫描等实验方法初步研究了该基因缺失对变形链球菌生物学特性的影响,为龋病防治提供了新的思路和新的方法。
Dental caries is one of the most common chronic diseases afflicting humans and has a polymicrobial etiology. Streptococcus mutans (S. mutans) is one of the human oral caries bacteria, as well as the prime pathogen agents in the development of dental caries. An important virulence property of S. mutans is to form dental plaque biofilm on the tooth surface. Bacteria in the biofilm grow, reproduce, metabolize and constitute a special micro ecological environment.
Comparative genomic study found a class of GGDEF/EAL domain protein widespread in various types of bacteria. Further studies showed that its main function is about synthesis and degradation of cyclic di(3'→5')-guanylic acid (c-di-GMP). The GGDEF/EAL domain could catalyze GTPs to c-di-GMP. The GGDEF and EAL domain families have1,601and1,016members, respectively, in the Pfam protein family database in July2005. Proteins containing those domains are involved in motility and exopolysaccharide-biofilm production in a variety of bacteria. Some GGDEF-domain proteins control biofilm formation and/or cell aggregation by enhancing the levels of the novel second messenger c-di-GMP. Intracellular concentrations of c-di-GMP are regulated by multiple domain proteins. C-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) proteins containing the amino acid motif GGDEF domain. C-di-GMP is degraded by phosphodiesterases (PDEs) that contain one of two conserved domain families:one defined by the EAL motif and the other by an HD-GYP motif. C-di-GMP plays an important role in the physiology of many bacteria.
Recent studies show that c-di-GMP is one of the second messager signaling molecules. It could regulate many processes to adapt to environment in bacteria. It involves in the biofilm formation, virulence, communication and the interaction between the host cells and bacteria. It is one of the key regulatory factors in bacterial survival and metabolism.
There is c-di-GMP signaling pathway in Streptococcus mutans. Adding exogenous c-di-GMP or reducing endogenous c-di-GMP will inhibit the biofilm formation of Streptococcus mutans. But the mechanisms are unknown. Further research on these issues will help to deepen the role of c-di-GMP signaling pathways in Streptococcus mutans and explore a new method of anticaries. Construct gcp gene mutation strains of Streptococcus mutans. Then extract the RNA of mutation strain and wild strain. Compare the differences by microarray chip and screen the biofilm-related genes to analysis the signaling passways.
This paper includes the following three chapters:
Chapter I:Constructed the gcp gene mutation strain of S. mutans UA159
Designed the upstream primers and downstream primers of gcp gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Constructed a vector containing spectinomycin resistance (spe+) pFW5-gcp-UP-DOWN. Constructed S. mutans UA159gcp gene mutation strain which was named gcp-KO by homologous recombination. Under an optical microscope, S. mutans UA159was G+, arranged in chains with different lengths. The gcp-KO strain was G+too, arranged in short chains and more sparse arrangement compared to the wild strain. This might be due to genetic defects. Gel electrophoresis was used to identify the upstream and downstream of gcp gene and the spectinomycin gene in wild strain and mutation strain. The results showed that the wild strain didn't contain the spectinomycin gene but contain the gcp gene. The mutation strain could amplify upstream and downstream of gcp gene and amplify the spectinomycin gene, but it couldn't amplify the gcp gene. Real-time PCR showed gcp gene a down-regulation of the gcp-KO strain. All the results showed the mutation strain was gcp gene defect strain. The sequencing results also showed that the gcp gene was replaced by the spectinomycin gene.
Chapter Ⅱ:Microarray chip experiments in gcp-KO strain and wild strain of S. mutans UA159
Extracted the RNA of wild strain and gcp-KO strain. Then synthesized "cDNA" by the instructions of "TaKaRa1st Strand cDNA Synthesis Kit" and marked the control group by Cy5-CTP and experimental group by Cy3-CTP. Immediately absorbed the appropriate amount of hybridization mixture on the slide and put the slide into NimbleGen MS200gene chip scanner. Laboratory assistant collected the data with MS200software.
Compared the Cy5fluorescence signal strength and Cy3signal strength of the two groups and got the ratio by Cy5than Cy3. For the same probe, if the ratio was2times or more than2times, gene expression of this probe was downregulated. If the ratio was0.5times or less than0.5times, gene expression of this probe was upregulated. Finally got411genes with198gene upregulated and213gene down regulated. After GO analysis, COG analysis and KEGG analysis, choose8genes for Real-time PCR. Think about the previous experiments results, RgpAc gene is selected as the target gene in the following studies.
Chapter III Construction and function studies of RgpAc mutation strain
This chapter includes two aspects:
"Construction of RgpAc gene mutation strain" and "Function studies of RgpAc gene mutation strain"
1. Construction of RgpAc gene mutation strain
Designed the upstream primers and downstream primers of RgpAc gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Then constructed a suicide vector containing spectinomycin resistance (spe+) pFW5-RgpAc-UP-DOWN. Finally construct S. mutans UA159RgpAc gene mutation strain which was named RgpAc-KO by homologous recombination. Gel electrophoresis was used to identify the upstream and downstream of RgpAc gene and the spectinomycin gene in wild strain and mutation strain. The experimental results showed that the wild strain contained the RgpAc gene, not the spectinomycin gene. The mutation strain could amplify upstream and downstream of RgpAc gene and the spectinomycin gene, but it couldn't amplify the RgpAc gene. Real-time PCR showed RgpAc gene downregulation in the RgpAc-KO strain. All the results showed the mutation strain was RgpAc gene defect strain.
2. Function studies of RgpAc gene mutation strain
This study compared the extracellular polysaccharide synthesis capacity of RgpAc gene mutation strain and wild strain by modified anthrone colorimetry. SPSS13.0statistical software was used to analysis the results. The formation of water-soluble glucan of RgpAc-KO strain decreased by55%, exopolysaccharide by41%. They were significantly lower than wild strain. But there was no significant difference in the ability to form water-insoluble glucan between RgpAc-KO strain and wild strain. The RgpAc gene was glycosyltransferase in S. mutans UA159. Glycosyltransferases catalyzed the transfer of sugar moieties to specific acceptor molecules and formed glycosidic bonds. This glycosyltransferase mainly relates to water-soluble dextran a-1,6. When RgpAc gene was knocked out, the ability of synthetic dextran a-1,6was inhibited and water-soluble glucan was decreased.
Designed the experiment about biofilm formation in different strains. RgpAc-KO strain was experimental group and wild strain was control group. Then added200μM and400μM c-di-GMP into wild strain and RgpAc-KO strain. Both strains grown aerobically at37℃in a5%CO2atmosphere under static conditions for48h. Measured the absorbance value at575nm. The biofilm formation of S. mutans UA159was inhibited after adding c-di-GMP to the culture. However, no major differences were observed between different concentrations of c-di-GMP. The results showed that the RgpAc-KO strain was less efficient55%at forming biofilm than wild strain. The biofilm formation of RgpAc-KO strain was promoted after adding c-di-GMP to the culture. In this study, the biofilm formation of RgpAc-KO was decreased. However, the biofilm formation of RgpAc-KO strain with200μM c-di-GMP and400μM c-di-GMP was increased. Water-soluble and water-insoluble glucan are the biofilm matrix. The ability of the water-soluble glucan decreased after RgpAc gene knocked out. The biofilm formation was inhibited because lack of matrix. When extracellular c-di-GMP was added in the RgpAc-KO strain, it increased the binding of other receptors and promoted the biofilm formation.
To observe the biofilm structure of RgpAc-KO strain with the c-di-GMP, prepared the enamel pieces and simulated the vivo situation to form artificial biofilm on the enamel pieces. Then biofilm structure was detected by a combination of scanning electron microscopy. Under low magnification, thick and compact biofilm was observed of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. However, thin and loose biofilm was observed of RgpAc-KO strain. Under high magnification, cells with large extracellular matrix arranged regularly of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. Cells with little extracellular matrix arranged irregularly of RgpAc-KO strain. RgpAc-KO strain reduced the synthesis of polysaccharide, especially glucan. Glucan and other polysaccharide were energy substances and metabolic substrates, as well as promoted bacteria adhesion and aggregation. When extracellular c-di-GMP was added in the RgpAc-KO strain, it played a role of endogenous second message and finally increased the biofilm formation. But RgpAc-KO strain reduced the addition of extracellular polysaccharide and suppressed the ability of biofilm synthesis. Under scanning electron microscope, loose extracellular matrix and less bacteria were observed.
In conclusion, this study constructs gcp gene knockout strain of Streptococcus mutans UA159by homologous recombination. Microarray analysis finds the RgpAc gene is upregulation. Then construct RgpAc gene knockout strain by the same way and design studies about its functions. The results will provide new methods and ideas for caries prevention.
比较基因组学研究发现一类含有GGDEF/EAL结构域的蛋白在各类细菌中普遍存在,进一步研究发现其功能主要是参与3'-5'环二鸟苷酸(bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP)的合成与分解,该结构域能够催化两个GTPs合成c-di-GMP。大部分的GGDEF结构域在临近N端的活性位点处含有一个Ⅰ位点,该位点可以与非竞争性抑制产物c-di-GMP结合,这种别构抑制作用能够限制c-di-GMP的浓度,阻止GTP的过度消耗。磷酸二酯酶(phosphodiesterases, PDEs)上的EAL结构域具有水解c-di-GMP的作用,其水解产物是线性的5'-磷酸鸟苷酸-(3’-5')-鸟苷(5'-phosphoguany1y1-(3'-5')-guanosine,5'-pGpG)分子,这些小分子物质不具备生物活性,在细胞内很快被其它的磷酸二酯酶降解。通常情况下,GGDEF和EAL结构域存在于同一个蛋白的C端,而负责信号感知及传导的结构域则位于蛋白的N端,可以感知一系列信号,例如PAS和GAF结构域,涉及到细胞内小分子的结合或者蛋白与蛋白之间的相互作用。除此之外,还有一小部分GGDEF和EAL结构域蛋白的N端整合在细胞膜上,负责感知信号,但是目前还不清楚这些结构域能够感知什么样的信号。
近年来的研究已经证实,c-di-GMP是原核细胞信号转导中一类新的第二信使,是一种小分子复合物,具有热稳定性,能够激活纤维素合成酶,参与多种复杂的生理活动的调控。它的作用涉及细菌细胞的分化、生物膜的形成、细胞间的通讯、毒力因子的表达以及与宿主细胞间相互作用等多个方面,是细菌生存和代谢过程中的关键性调节因子之一。
研究发现变形链球菌中存在c-di-GMP信号通路,外源性c-di-GMP的加入和内源性c-di-GMP的丧失均会降低变形链球菌的致龋特性(表现为生物膜形成量减少),但其机制尚不清楚。对这些问题进一步的研究将有助于加深对变形链球菌中c-di-GMP信号途径的作用方式的认识,也为探索新的防龋方法提供了依据。本课题组在前期研究的基础上,构建了变形链球菌内gcp基因敲除菌株,以便更全面、深入的探讨变形链球菌内c-di-GMP的作用机制和信号通路,同时与S. mutans UA159野生菌株进行基因芯片比较,筛选和生物膜有关的基因进行信号通路分析。
本论文包括以下三部分内容：
第一章：变形链球菌gcp基因突变株的构建
本实验根据NCBI提供的S. mutans UA159全基因组序列,采用Primer Premier5.0软件(Premier公司)设计gcp基因的上游引物和下游引物,扩增gcp基因的上游片段和下游片段。采用双酶切及同源重组的方式构建了含有壮观霉素抗性(spe)的载体pFW5-gcp-UP-DOWN。在下一步实验中,采用同源重组的方式构建变形链球菌gcp基因突变菌株。
用限制性内切酶Nhe Ⅰ酶(NEB公司)对载体pFW5-gcp-UP-DOWN进行酶切,将环状的质粒变为线性化的片段,胶回收酶切后的线性化片段10ug备用。取1ml变形链球菌感受态细胞菌液,加入30μl线性化的重组载体pFW5-gcp-UP-DOWN,采用同源重组的方式构建变形链球菌gcp基因突变菌株,命名为gcp-KO。
通过对转化菌的形态学观察发现,转化菌形态与S. mutans UA159菌基本一致,但是细菌排列更为稀疏,这可能是由于基因缺陷后引起的生长差异所致。对重组载体进行双酶切鉴定结果显示：重组载体pFW5-gcp-UP经过Sal Ⅰ和Bgl Ⅱ双酶切后出现2条带,与质粒pFW5-gcp-UP的大小基本相同。重组载体pFW5-gcp-UP-DOWN经过Nco Ⅰ和Nde Ⅰ双酶切后出现2条带,与质粒pFW5-gcp-UP及gcp-DOWN的大小基本相同。对野生菌与转化菌的gcp基因上下游片段、spe基因进行PCR扩增及电泳鉴定结果显示：野生菌株不能扩增spe基因,而转化菌株能够扩增上、下游基因及spe基因,表明spe基因成功转入菌株基因组,测序结果也显示gcp基因被spe基因所同源替代。
第二章：变形链球菌gcp基因突变菌株表达谱基因芯片研究及验证
提取变形链球菌gcp-KO及野生菌株的总RNA,参照大连宝生物工程公司1st Strand cDNA Synthesis Kit试剂盒说明书分别合成cDNA。对照组(野生菌株)加入Cy5-CTP*(10mmol/L),实验组(突变菌株)加入Cy3-CTP*(10mmol/L)进行标记,并采用3S柱PCR产物纯化试剂盒纯化,得到杂交混合液。吸取适量杂交混合液,使样品液均匀的覆盖在玻片的表面,检查气泡后将基因芯片字符面朝上放入芯片夹中,将芯片夹置入基因芯片扫描仪NimbleGen MS200中,采用MS200Data Collection软件,由仪器自动平衡红绿荧光的激发光能量及增益值进行扫描,获得双色叠加的杂交结果图,保存该结果。
本实验为了确定差异基因表达谱,采用国内外基因芯片实验常用标准Ratio'=2做为差异基因的初步筛选标准。上调基因筛选的转录差异要在2倍以上,即FC≥2。同样下调基因筛选的转录差异也是2倍以上,即FC≤0.5。转录差异在0.5≤FC≤2之间的基因,被认为没有差别,不筛选进入下一步研究,最终有411个基因符合条件。分别比较两组数据中Cy5荧光信号强度和Cy3荧光信号强度,对于同一个探针,如果Cy5信号强度是Cy3的2倍或2倍以上,认为该探针所代表的基因表达降低；如果Cy3的信号强度是Cy5的2倍或2倍以上,说明该探针代表的基因表达升高。初步筛选411个差异基因,表达上调的基因198个,表达下调的基因213个。再结合GO分析、COG分析及KEGG分析的结果,最终确定8个基因进入本课题的Real-time PCR验证研究,其中上调基因和下调基因各4个。表达差异的基因按照功能进行归类,查阅GenBank数据库及相关文献对其结果进行深入的分析及讨论。结合前期实验的结果,选择RgpAc基因作为后续研究的目的基因。
第三章：RgpAc基因突变株的构建及功能研究
本章主要包括两方面的内容：
1.RgpAc基因突变株的构建
本实验根据NCBI提供的S. mutans UA159全基因组序列,采用Primer Premier5.0软件设计RgpAc基因的上游引物和下游引物,扩增RgpAc基因的上游片段和下游片段。采用双酶切及同源重组的方式构建了含有壮观霉素抗性(spe)的载体pFW5-RgpAc-UP-DOWN。将载体加入变形链球菌感受态细胞中,采用同源重组的方式构建变形链球菌RgpAc基因突变菌株,命名为RgpAc-KO。通过对转化菌的形态学观察发现,转化菌形态与S. mutans UA159菌基本一致,但是细菌排列更为稀疏,这可能是由于基因缺陷后引起的生长差异所致。实验还对野生菌与转化菌的RgpAc基因及其上下游、spe基因进行了PCR扩增与电泳鉴定。结果显示,野生菌株含有RgpAc基因,不含有spe基因。而转化菌株能够扩增载体的上、下游基因及spe基因,不能扩增RgpAc基因,表明转化菌株有RgpAc基因缺陷。测序结果也显示RgpAc基因被spe基因所同源替代。
2.变形链球菌RgpAc基因功能的研究
采用SPSS13.0统计学软件进行统计学处理,对比了野生菌株和突变菌株在24h、48h形成的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖的含量,以及野生菌株和突变菌株加入不同浓度的c-di-GMP之后,形成的生物膜含量。采用析因设计的方差分析的方法,各组计量资料数据采用均数±标准差的方式列出,检验水准a=0.05。
通过改良蒽酮比色法比较突变菌株和野生菌株胞外多糖的合成能力。实验分为两组,分别为野生菌株和突变菌株,24h的样本量为8例,48h的样本量为5例。野生菌株水溶性葡聚糖和胞外多糖生成量高于突变菌株,差异有统计学意义(F=328.868, P<0.001; F=80.897, P<0.001),突变菌株水不溶性葡聚糖生成量高于野生菌株,差异有统计学意义(F=15.718,P<0.001)。
同一菌株不同的时间段相比,野生菌株24h和48h产生的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖差异均有统计学意义(F=36.022,P<0.001；F=15.274, P<0.001; F=-18.241, P<0.001),24h处产生的水溶性葡聚糖高于48h处,水不溶性葡聚糖低于48h处,胞外多糖高于48h处。突变菌株24h和48h产生的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖差异均有统计学意义(F=-7.537, P<0.001; F=18.762, P<0.001; F=-8.587, P<0.001),24h产生的水溶性葡聚糖高于48h处,水不溶性葡聚糖低于48h处,胞外多糖低于48h处。
在比较生物膜形成量的实验中,S.mutans UA159与RgpAc-KO加入200μM和400μM的c-di-GMP后,设计了6组,每组有5例,采用析因分析结果发现,两组菌株之间的差异有统计学意义(F=29.411,P<0.001),突变菌株高于野生菌株；三个c-di-GMP浓度间的差异有统计学意义(F=3.945, P=0.033),400μM组最高,空白对照组最低；在不加c-di-GMP干扰的时候,野生菌株和突变菌株之间的差异有统计学意义(t=8.196,P<0.001),野生菌株更高；在加入200μM、400μM的c-di-GMP干扰后,野生菌株和突变菌株之间的差异有统计学意义(t=-4.467, P=0.002;t=-8.038, P<0.001),突变菌株均高于野生菌株；野生菌株加入不同浓度的c-di-GMP干扰后,产生的差异有统计学意义(F=24.766,P<0.001),不加c-di-GMP组最高,加入400μM的c-di-GMP组最低；突变菌株加入不同浓度c-di-GMP干扰后,产生的差异有统计学意义(F=48.322,P<0.001),不加c-di-GMP组最低,加400μM的c-di-GMP组最高;交互效应有统计学意义(F=47.529,P<0.001)。实验结果表明变形链球菌RgpAc基因敲除后,细菌形成生物膜的能力被抑制。在变形链球菌中,各种水溶性和水不溶性的多糖是生物膜形成的重要基质之一。当变形链球菌的RgpAc基因敲除后,合成水溶性葡聚糖和胞外多糖的能力均下降,导致生物膜形成的基质缺乏,最终抑制了生物膜的形成。突变菌株加入不同浓度的c-di-GMP之后,生物膜形成能力增加,推测原因是外源性的c-di-GMP增加后促进了其与下游信号通路受体的结合,最终使生物膜的形成量增加。
为了比较两种细菌在牙齿表面形成的生物膜结构的差异,实验模拟体内情况在釉质表面形成人工生物膜,并进行了扫描电镜观察。结果显示,RgpAc-KO与野生菌株培养48h后,产生的生物膜形态不相同。低倍镜下可见S. mutansUA159组产生的生物膜较厚,排列较致密,RgpAc-KO组的生物膜结构较松散,突变菌株分别加入200μM和400μM的c-di-GMP之后,细胞外基质增加。高倍视野下可见S. mutans UA159组细菌之间有较多的细胞外基质,生物膜上的细菌排列规律,形成漩涡状结构；RgpAc-KO组细菌之间的细胞外基质较少,视野内可见少量的细菌。突变菌株分别加入200μM和400μM的c-di-GMP之后,细菌生物膜的结构变致密,细胞数目增多。提示RgpAc基因突变后降低了水溶性葡聚糖的合成,而水溶性葡聚糖既可作为细菌胞外能源物质及代谢底物,又可与细菌表面的Gbp结合,能够促进黏附和聚集。因此在电镜下观察突变菌株的生物膜结构较松散,细胞外基质较少,视野内细菌数量少。加入外源性c-di-GMP后增加了下游信号通路的活性,最终使生物膜的形成增加。
综上,本实验通过同源重组的方法构建了变形链球菌gcp基因突变菌株,与野生菌株进行了基因芯片比较,表达差异在2倍以上的基因有411个,包括198个表达上调的基因和213个表达下调的基因。通过查阅资料,最终挑选8个最有可能与变形链球菌生物膜形成及信号通路有关的基因进行验证,发现RgpAc基因表达抑制的程度最大,因此课题组选择该基因作为下一步研究的目的基因,构建了变形链球菌RgpAc基因突变菌株。利用比色、电镜扫描等实验方法初步研究了该基因缺失对变形链球菌生物学特性的影响,为龋病防治提供了新的思路和新的方法。
Dental caries is one of the most common chronic diseases afflicting humans and has a polymicrobial etiology. Streptococcus mutans (S. mutans) is one of the human oral caries bacteria, as well as the prime pathogen agents in the development of dental caries. An important virulence property of S. mutans is to form dental plaque biofilm on the tooth surface. Bacteria in the biofilm grow, reproduce, metabolize and constitute a special micro ecological environment.
Comparative genomic study found a class of GGDEF/EAL domain protein widespread in various types of bacteria. Further studies showed that its main function is about synthesis and degradation of cyclic di(3'→5')-guanylic acid (c-di-GMP). The GGDEF/EAL domain could catalyze GTPs to c-di-GMP. The GGDEF and EAL domain families have1,601and1,016members, respectively, in the Pfam protein family database in July2005. Proteins containing those domains are involved in motility and exopolysaccharide-biofilm production in a variety of bacteria. Some GGDEF-domain proteins control biofilm formation and/or cell aggregation by enhancing the levels of the novel second messenger c-di-GMP. Intracellular concentrations of c-di-GMP are regulated by multiple domain proteins. C-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) proteins containing the amino acid motif GGDEF domain. C-di-GMP is degraded by phosphodiesterases (PDEs) that contain one of two conserved domain families:one defined by the EAL motif and the other by an HD-GYP motif. C-di-GMP plays an important role in the physiology of many bacteria.
Recent studies show that c-di-GMP is one of the second messager signaling molecules. It could regulate many processes to adapt to environment in bacteria. It involves in the biofilm formation, virulence, communication and the interaction between the host cells and bacteria. It is one of the key regulatory factors in bacterial survival and metabolism.
There is c-di-GMP signaling pathway in Streptococcus mutans. Adding exogenous c-di-GMP or reducing endogenous c-di-GMP will inhibit the biofilm formation of Streptococcus mutans. But the mechanisms are unknown. Further research on these issues will help to deepen the role of c-di-GMP signaling pathways in Streptococcus mutans and explore a new method of anticaries. Construct gcp gene mutation strains of Streptococcus mutans. Then extract the RNA of mutation strain and wild strain. Compare the differences by microarray chip and screen the biofilm-related genes to analysis the signaling passways.
This paper includes the following three chapters:
Chapter I:Constructed the gcp gene mutation strain of S. mutans UA159
Designed the upstream primers and downstream primers of gcp gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Constructed a vector containing spectinomycin resistance (spe+) pFW5-gcp-UP-DOWN. Constructed S. mutans UA159gcp gene mutation strain which was named gcp-KO by homologous recombination. Under an optical microscope, S. mutans UA159was G+, arranged in chains with different lengths. The gcp-KO strain was G+too, arranged in short chains and more sparse arrangement compared to the wild strain. This might be due to genetic defects. Gel electrophoresis was used to identify the upstream and downstream of gcp gene and the spectinomycin gene in wild strain and mutation strain. The results showed that the wild strain didn't contain the spectinomycin gene but contain the gcp gene. The mutation strain could amplify upstream and downstream of gcp gene and amplify the spectinomycin gene, but it couldn't amplify the gcp gene. Real-time PCR showed gcp gene a down-regulation of the gcp-KO strain. All the results showed the mutation strain was gcp gene defect strain. The sequencing results also showed that the gcp gene was replaced by the spectinomycin gene.
Chapter Ⅱ:Microarray chip experiments in gcp-KO strain and wild strain of S. mutans UA159
Extracted the RNA of wild strain and gcp-KO strain. Then synthesized "cDNA" by the instructions of "TaKaRa1st Strand cDNA Synthesis Kit" and marked the control group by Cy5-CTP and experimental group by Cy3-CTP. Immediately absorbed the appropriate amount of hybridization mixture on the slide and put the slide into NimbleGen MS200gene chip scanner. Laboratory assistant collected the data with MS200software.
Compared the Cy5fluorescence signal strength and Cy3signal strength of the two groups and got the ratio by Cy5than Cy3. For the same probe, if the ratio was2times or more than2times, gene expression of this probe was downregulated. If the ratio was0.5times or less than0.5times, gene expression of this probe was upregulated. Finally got411genes with198gene upregulated and213gene down regulated. After GO analysis, COG analysis and KEGG analysis, choose8genes for Real-time PCR. Think about the previous experiments results, RgpAc gene is selected as the target gene in the following studies.
Chapter III Construction and function studies of RgpAc mutation strain
This chapter includes two aspects:
"Construction of RgpAc gene mutation strain" and "Function studies of RgpAc gene mutation strain"
1. Construction of RgpAc gene mutation strain
Designed the upstream primers and downstream primers of RgpAc gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Then constructed a suicide vector containing spectinomycin resistance (spe+) pFW5-RgpAc-UP-DOWN. Finally construct S. mutans UA159RgpAc gene mutation strain which was named RgpAc-KO by homologous recombination. Gel electrophoresis was used to identify the upstream and downstream of RgpAc gene and the spectinomycin gene in wild strain and mutation strain. The experimental results showed that the wild strain contained the RgpAc gene, not the spectinomycin gene. The mutation strain could amplify upstream and downstream of RgpAc gene and the spectinomycin gene, but it couldn't amplify the RgpAc gene. Real-time PCR showed RgpAc gene downregulation in the RgpAc-KO strain. All the results showed the mutation strain was RgpAc gene defect strain.
2. Function studies of RgpAc gene mutation strain
This study compared the extracellular polysaccharide synthesis capacity of RgpAc gene mutation strain and wild strain by modified anthrone colorimetry. SPSS13.0statistical software was used to analysis the results. The formation of water-soluble glucan of RgpAc-KO strain decreased by55%, exopolysaccharide by41%. They were significantly lower than wild strain. But there was no significant difference in the ability to form water-insoluble glucan between RgpAc-KO strain and wild strain. The RgpAc gene was glycosyltransferase in S. mutans UA159. Glycosyltransferases catalyzed the transfer of sugar moieties to specific acceptor molecules and formed glycosidic bonds. This glycosyltransferase mainly relates to water-soluble dextran a-1,6. When RgpAc gene was knocked out, the ability of synthetic dextran a-1,6was inhibited and water-soluble glucan was decreased.
Designed the experiment about biofilm formation in different strains. RgpAc-KO strain was experimental group and wild strain was control group. Then added200μM and400μM c-di-GMP into wild strain and RgpAc-KO strain. Both strains grown aerobically at37℃in a5%CO2atmosphere under static conditions for48h. Measured the absorbance value at575nm. The biofilm formation of S. mutans UA159was inhibited after adding c-di-GMP to the culture. However, no major differences were observed between different concentrations of c-di-GMP. The results showed that the RgpAc-KO strain was less efficient55%at forming biofilm than wild strain. The biofilm formation of RgpAc-KO strain was promoted after adding c-di-GMP to the culture. In this study, the biofilm formation of RgpAc-KO was decreased. However, the biofilm formation of RgpAc-KO strain with200μM c-di-GMP and400μM c-di-GMP was increased. Water-soluble and water-insoluble glucan are the biofilm matrix. The ability of the water-soluble glucan decreased after RgpAc gene knocked out. The biofilm formation was inhibited because lack of matrix. When extracellular c-di-GMP was added in the RgpAc-KO strain, it increased the binding of other receptors and promoted the biofilm formation.
To observe the biofilm structure of RgpAc-KO strain with the c-di-GMP, prepared the enamel pieces and simulated the vivo situation to form artificial biofilm on the enamel pieces. Then biofilm structure was detected by a combination of scanning electron microscopy. Under low magnification, thick and compact biofilm was observed of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. However, thin and loose biofilm was observed of RgpAc-KO strain. Under high magnification, cells with large extracellular matrix arranged regularly of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. Cells with little extracellular matrix arranged irregularly of RgpAc-KO strain. RgpAc-KO strain reduced the synthesis of polysaccharide, especially glucan. Glucan and other polysaccharide were energy substances and metabolic substrates, as well as promoted bacteria adhesion and aggregation. When extracellular c-di-GMP was added in the RgpAc-KO strain, it played a role of endogenous second message and finally increased the biofilm formation. But RgpAc-KO strain reduced the addition of extracellular polysaccharide and suppressed the ability of biofilm synthesis. Under scanning electron microscope, loose extracellular matrix and less bacteria were observed.
In conclusion, this study constructs gcp gene knockout strain of Streptococcus mutans UA159by homologous recombination. Microarray analysis finds the RgpAc gene is upregulation. Then construct RgpAc gene knockout strain by the same way and design studies about its functions. The results will provide new methods and ideas for caries prevention.
引文
[1]Sudhir R, Praveen P, Anantharaj A, et al. Assessment of the effect of probiotic curd consumption on salivary pH and streptococcus mutans counts [J]. Niger Med J,2012,53(3):135-139.
[2]Ko J, Ryu KS, Kim H, et al. Structure of PP4397 reveals the molecular basis for different c-di-GMP binding modes by Pilz domain proteins [J]. J Mol Biol,2010, 398(1):97-110.
[3]Kozlova EV, Khajanchi BK, Sha J, et al. Quorum sensing and c-di-GMP-dependent alterations in gene transcripts and virulence-associated phenotypes in a clinical isolate of Aeromonas hydrophila [J]. Microb Pathog, 2011,50(5):213-223.
[4]Amikam D, Galperin MY. PilZ domain is part of the bacterial c-di-GMP binding protein [J]. Bioinformatics,2006,22(1):3-6.
[5]Lory S, Merighi M, Hyodo M. Multiple activities of c-di-GMP in Pseudomonas aeruginosa [J]. Nucleic Acids Symp Ser,2009,53(1):51-52.
[6]Newell PD, Monds RD, O'Toole GA. LapD is a bis-(3',5')-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1 [J]. Proc Natl Acad Sci USA,2009,106(9):3461-3466.
[7]Ueda A, Wood TK. Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885) [J]. PLoS Pathog,2009,5(6):e1000483.
[8]Karaolis DK, Rashid MH, Chythanya R, et al. c-di-GMP (3'-5'-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation [J]. Antimicrob Agents Chemother,2005,49(3):1029-1038.
[9]Frederick JR, Sarkar J, McDowell JV, et al. Molecular signaling mechanisms of the periopathogen, Treponema denticola [J]. J Dent Res,2011,90(10):1155-1163.
[10]Schirmer T, Jenal U. Structural and mechanistic determinants of c-di-GMP signalling [J]. Nat Rev Microbiol,2009,7(10):724-735.
[11]Yan W, Qu T, Zhao H, et al. The effect of c-di-GMP (3'-5'-cyclic diguanylic acid) on the biofilm formation and adherence of Streptococcus mutans [J]. Microbiol Res,2010,165(2):87-96.
[12]闫文娟,吴补领,屈铁军,等.外源性c-di-GMP对变形链球菌产酸、耐酸能力的影响[J].牙体牙髓牙周病学杂志,2009,19(9)：510-512.
[13]Dong C, Zhang FQ. Effect of denture base materials on mRNA expression of the adhesion-associated genes from the Streptococcus mutans biofilms [J]. J Oral Rehabil,2009,36(12):894-901.
[14]Wen ZT, Burne RA. LuxS-mediated signaling in Streptococcus mutans is involved in regulation of acid and oxidative stress tolerance and biofilm formation [J]. J Bacteriol,2004,186(9):2682-2691.
[15]Wen ZT, Nguyen AH, Bitoun JP, et al. Transcriptome analysis of LuxS-deficient Streptococcus mutans grown in biofilms [J]. Mol Oral Microbiol,2011, 26(1):2-18.
[16]Shemesh M, Tam A, Kott-Gutkowski M, et al. DNA-microarrays identification of Streptococcus mutans genes associated with biofilm thickness [J]. BMC Microbiol,2008,8:236.
[17]Nguyen T, Zhang Z, Huang IH, et al. Genes involved in the repression of mutacin I production in Streptococcus mutans [J]. Microbiology,2009,155(2):551-556.
[18]闫文娟,徐树军,谢苗苗,等.外源性单磷酸乌苷环二聚体对变形链球菌基因表达的影响[J].中国组织工程研究,2012,16(8)：1451-1454.
[19]Galperin MY, Nikolskaya AN, Koonin EV. Novel domains of the prokaryotic two-component signal transduction systems [J]. FEMS Microbiol Lett,2001, 203:11-21.
[20]Ross P, Weinhouse H, Aloni Y, et al. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid [J]. Nature,1987,325(6101): 279-281.
[21]Amikam D, Benziman M. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens [J]. J Bbacteriol,1989,171(12):6649-6655.
[22]Srivastava D, Waters CM. A tangled web:regulatory connections between quorum sensing and cyclic Di-GMP [J]. Journal of bacteriology,2012,194(17): 4485-4493.
[23]Sintim HO, Smith JA, Wang J, et al. Paradigm shift in discovering next-generation anti-infective agents_ targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules [J]. Future Med Chem,2010,2(6):1005-1035.
[24]Shanahan CA, Strobel SA. The bacterial second messenger c-di-GMP:probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs [J]. Org Biomol Chem,2012,10(46):9113-9129.
[25]Tchigvintsev A, Xu X, Singer A, et al. Structural insight into the mechanism of cyclic di-GMP hydrolysis by EAL domain phosphodiesterases [J]. J Mol Biol, 2010,402(24):524-538.
[26]Jenal U. Cyclic di-guanosine-monophosphate comes of age:a novel secondary messenger involved in modulating cell surface structures in bacteria? [J]. Curr Opin Microbiol,2004,7:185-191.
[27]Boyd CD, O'Toole GA. Second messenger regulation of biofilm formation: breakthroughs in understanding c-di-GMP effector systems [J]. Annu Rev Cell Dev Biol,2012,28:439-462.
[28]Rao F, Pasunooti S, Ng Y, et al. Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase [J]. Anal Biochem,2009,389(15):138-142.
[29]Navarro MV, De N, Bae N, et al. Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX [J]. Structure,2009, 17(8):1104-1116.
[30]Bordeleau E, Fortier LC, Malouin F, et al. c-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases [J]. PLoS Genet,2011,7(3):e1002039.
[31]Romling U, Gomelsky M, Galperin MY. C-di-GMP:the dawning of a novel bacterial signalling system [J]. Mol Microbiol,2005,57(3):629-639.
[32]Sondermann H, Shikuma NJ, Yildiz FH. You've come a long way:c-di-GMP signaling [J]. Current opinion in microbiology,2012,15(2):140-146.
[33]Jonas K, Melefors O, Romling U. Regulation of c-di-GMP metabolism in biofilms [J]. Future Microbiol,2009,4(3):341-358.
[34]Ryan RP, Dow JM. Intermolecular interactions between HD-GYP and GGDEF domain proteins mediate virulence-related signal transduction in Xanthomonas campestris [J]. Virulence,2010, 1(5):404-408.
[35]Galperin MY. A census of membrane-bound and intracellular signal transduction proteins in bacteria:bacterial IQ, extroverts and introverts [J]. BMC Microbiol, 2005,5:35.
[36]Yildiz FH, Visick KL. Vibrio biofilms:so much the same yet so different [J]. Trends Microbiol,2009,17(3):109-118.
[37]Nakhamchik A, Wilde C, Rowe-Magnus DA. Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus [J]. Appl Environ Microbiol,2008, 74(13):4199-4209.
[38]Krasteva PV, Fong JC, Shikuma NJ, et al. Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP [J]. Science,2010, 327(5967):866-868.
[39]Sun S, Kjelleberg S, McDougald D. Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists [J]. PloS One,2013,8(2):e56338.
[40]Tischler AD, Camilli A. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation [J]. Mol Microbiol,2004,53(3):857-869.
[41]Pratt JT, McDonough E, Camilli A. PhoB regulates motility, biofilms, and cyclic di-GMP in Vibrio cholerae [J]. J Bacteriol,2009,191(21):6632-6642.
[42]Li TN, Chin KH, Fung KM, et al. A Novel Tetrameric PilZ Domain Structure from Xanthomonads [J]. PloS One,2011,6(7):e22036.
[43]Whitney JC, Colvin KM, Marmont LS, et al. Structure of the cytoplasmic region of PelD, a degenerate diguanylate cyclase receptor that regulates exopolysaccharide production in Pseudomonas aeruginosa [J]. J Biol Chem,2012, 287(28):23582-23593.
[44]Jimenez PN, Koch G, Thompson JA, et al. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa [J]. Microbiol Mol Biol Rev, 2012,76(1):46-65.
[45]Lee VT, Matewish JM, Kessler JL, et al. A cyclic-di-GMP receptor required for bacterial exopolysaccharide production [J]. Mol Microbiol,2007, 65(6):1474-1484.
[46]Wolfe AJ, Visick KL. Get the message out:cyclic-Di-GMP regulates multiple levels of flagellum-based motility [J]. J Bacteriol,2008,190(2):463-475.
[47]Hay ID, Remminghorst U, Rehm BH. MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa [J]. Appl Environ Microbiol,2009,75(4):1110-1120.
[48]Jain R, Behrens AJ, Kaever V, et al. Type IV pilus assembly in Pseudomonas aeruginosa over a broad range of cyclic di-GMP concentrations [J]. J Bacteriol, 2012,194(16):4285-4294.
[49]Coggan KA, Wolfgang MC. Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype [J]. Curr Issues Mol Biol,2012,14(2):47-70.
[50]Guo Y, Rowe-Magnus DA. Identification of a c-di-GMP-regulated polysaccharide locus governing stress resistance and biofilm and rugose colony formation in Vibrio vulnificus [J]. Infect Immun,2010,78(3):1390-1402.
[51]Abel S, Chien P, Wassmann P, et al. Regulatory cohesion of cell cycle and cell differentiation through interlinked phosphorylation and second messenger networks [J]. Mol Cell,2011,43(4):550-560.
[52]Sultan SZ, Pitzer JE, Boquoi T, et al. Analysis of the HD-GYP domain cyclic dimeric GMP phosphodiesterase reveals a role in motility and the enzootic life cycle of Borrelia burgdorferi [J]. Infect Immun,2011,79(8):3273-3283.
[53]Chen MW, Kotaka M, Vonrhein C, et al. Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling [J]. J Bacteriol,2012,194(18):4837-4846.
[54]Roy AB, Petrova OE, Sauer K. The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion [J]. J Bacteriol,2012, 194(11):2904-2915.
[55]Sun YC, Koumoutsi A, Jarrett C, et al. Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases [J]. PloS One,2011,6(4):e19267.
[56]Kirillina O, Fetherston JD, Bobrov AG, et al. HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis [J]. Mol Microbiol,2004,54:75-88.
[57]Hickman JW, Tifrea DF, Harwood CS. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels [J]. Proc Natl Acad Sci USA,2005,102:14422-14427.
[58]Purcell EB, McKee RW, McBride SM, et al. Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile [J]. J Bacteriol,2012, 194(13):3307-3316.
[59]Wang KC, Hsu YH, Huang YN, et al. A previously uncharacterized gene stm0551 plays a repressive role in the regulation of type 1 fimbriae in Salmonella enterica serotype Typhimurium [J]. BMC microbiol,2012,12:111.
[60]Wang H, Wu JH, Ayala JC, et al. Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease [J]. J Bacteriol,2011,193(23):6529-6538.
[61]Mikami Y, Suzuki N, Takahashi T, et al. Bacitracin Upregulates mbrAB Transcription via mbrCD to Confer Bacitracin Resistance in Streptococcus mutans [J]. J Pharmacol Sci,2011,117(3):204-207.
[62]Beyhan S, Tischler AD, Camilli A, et al. Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di-GMP level [J]. J Bacteriol,2006, 188(10):3600-3613.
[63]Kajfasz JK, Martinez AR, Rivera-Ramos I, et al. Role of Clp proteins in expression of virulence properties of Streptococcus mutans [J]. J Bacteriol,2009, 191(7):2060-2068.
[64]Trimble MJ, McCarter LL. Bis-(3'-5')-cyclic dimeric GMP-linked quorum sensing controls swarming in Vibrio parahaemolyticus [J]. Proc Natl Acad Sci USA,2011,108(44):18079-18084.
[65]Chong P, Chattoraj P, Biswsa I. Activation of the SMU.1882 Transcription by CovR in Streptococcus mutans [J]. PloS One,2012,5(11):e15528.
[66]Shemesh M, Tam A, Aharoni R, et al. Genetic adaptation of Streptococcus mutans during biofilm formation on different types of surfaces [J]. BMC Microbiol,2010,10:51.
[67]Lim B, Beyhan S, Yildiz FH. Regulation of Vibrio polysaccharide synthesis and virulence factor production by CdgC, a GGDEF-EAL domain protein, in Vibrio cholerae [J]. J Bacteriol,2007,189(3):717-729.
[68][68] Shikuma NJ, Fong JC, Yildiz FH. Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT [J]. PLoS pathogens,2012,8(5):e 1002719.
[69]Ryan RP, McCarthy Y, Andrade M, et al. Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris [J]. Proc Natl Acad Sci USA,2010, 107(13):5989-5994.
[70]Ahmad I, Lamprokostopoulou A, Le Guyon S, et al. Complex c-di-GMP Signaling Networks Mediate Transition between Virulence Properties and Biofilm Formation in Salmonella enterica Serovar Typhimurium [J]. PloS One, 2011,6(12):e28351.
[71]Hisert KB, MacCoss M, Shiloh MU, et al. A glutamatealanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages:role of cyclic diGMP [J]. Mol Microbiol, 2005,56(5):1234-45.
[72]Chung IY, Choi KB, Heo YJ, et al. Effect of PEL exopolysaccharide on the wspF mutant phenotypes in Pseudomonas aeruginosa PA14 [J]. J Microbiol Biotechnol, 2008,18(7):1227-1234.
[73]Kulasakara H, Lee V, Brencic A, et al. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence [J]. Proc Natl Acad Sci USA,2006, 103(8):2839-44.
[74]Deng Y, Schmid N, Wang C, et al. Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover [J]. Proc Natl Acad Sci USA,2012, 109(38):15479-15484.
[75]Petters T, Zhang X, Nesper J, et al. The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus [J]. Mol Microbiol,2012,84(1):147-165.
[76]Romling U, Amikam D. C-di-GMP is a ubiquitous second messenger that controls the transition from a free-living, motile lifestyle to a biofilm lifestyle in many bacteria [J]. Curr Opin Microbiol,2006,9(2):218-228.
[77]Navarro MV, Newell PD, Krasteva PV, et al. Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis [J]. PLoS Biol,2011,9(2):e1000588.
[78]Yang CY, Chin KH, Chuah ML, et al. The structure and inhibition of a GGDEF diguanylate cyclase complexed with (c-di-GMP)(2) at the active site [J]. Acta Crystallogr D Biol Crystallogr,2011,67(12):997-1008.
[79]Benach J, Swaminathan SS, Tamayo R, et al. The structural basis of cyclic diguanylate signal transduction by PilZ domains [J]. EMBO J,2007, 26(24):5153-5166.
[80]Fang X, Gomelsky M. A post-translational, c-di-GMP-dependent mechanism regulating flagellar motility [J]. Mol Microbiol,2010,76:1295-1305.
[81]Srivastava D, Harris RC, Waters CM. Integration of cyclic di-GMP and quorum sensing in the control of vpsT and aphA in Vibrio cholerae [J]. J Bacteriol,2011, 193(22):6331-6341.
[82]Rehman ZU, Rehm BH. Dual Roles of Pseudomonas aeruginosa AlgE in Secretion of the Virulence Factor Alginate and Formation of the Secretion Complex [J]. Appl Environ Microbiol,2013,79(6):2002-2011.
[83]Leduc JL, Roberts GP. Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri. [J]. J Bacteriol,2009, 191:7121-7122.
[84]Baird NJ, Kulshina N, Ferre-D'Amare AR. Riboswitch function:flipping the switch or tuning the dimmer? [J]. RNA Biol,2010,7(3):328-332.
[85]Beyhan S, Odelland LS, Yildiz FH. Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae [J]. J Bacteriol,2008,190:7392-7405.
[86]Liberman JA, Wedekind JE. Base ionization and ligand binding:how small ribozymes and riboswitches gain a foothold in a protein world [J]. Curr Opin Struct Biol,2011,21 (3):327-334.
[87]Petrova OE, Sauer K. Dispersion by Pseudomonas aeruginosa requires an unusual posttranslational modification of Bd1A [J]. Proc Natl Acad Sci USA, 2012,109(41):16690-16695.
[88]Massiea JP, Reynoldsa EL, Koestlera BJ, et al. Quantification of high-specificity cyclic diguanylate signaling [J]. Proc Natl Acad Sci USA,2012, 109(31):12746-12751.
[89]Kim JS, Shin DH. Inhibitory effect on Streptococcus mutans and mechanical properties of the chitosan containing composite resin [J]. Restor Dent Endod, 2013,38(1):36-42.
[90]Mohamed Hamouda I. Current perspectives of nanoparticles in medical and dental biomaterials [J]. J Biomed Res,2012,26(3):143-151.
[91]闫文娟,杨德鸿,吴补领.外源性单磷酸鸟苷环二聚体防龋的动物实验研究[J].南方医科大学学报,2012,32(5)：639-642.
[92]闫文娟.c-di-GMP信号通路在变形链球菌致龋过程中作用的初步研究[J]. 第四军医大学,2009：73-76.
[93]Ajdic D, McShan WM, McLaughlin RE, et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen [J]. Proc Natl Acad Sci USA,2002,99(22):14434-14439.
[94]Zhao L, KuoLee R, Harris G, et al. c-di-GMP protects against intranasal Acinetobacter baumannii infection in mice by chemokine induction and enhanced neutrophil recruitment [J]. Int Immunopharmacol,2011, 11(9):1378-1383.
[95]Koyanagi S, LevesqueCM. Characterization of a Streptococcus mutans Intergenic Region Containing a Small Toxic Peptide and Its cis-Encoded Antisense Small RNA Antitoxin [J]. PloS One,2012,8(1):e54291.
[96]Madhun AS, Haaheim LR, Nostbakken JK, et al. Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Thl CD4+ cells and strong mucosal and systemic antibody responses in mice [J]. Vaccine,2011,29(31):4973-4982.
[97]Costa MT, Dorta ML, Ribeiro-Dias F, et al. Biofilms of black tooth stains:PCR analysis reveals presence of Streptococcus mutans [J]. Braz Dent J,2012, 23(5):555-558.
[98]闫文娟.外源性单磷酸鸟苷环二聚体抑制变形链球菌生物膜的形成能力[J].中国组织工程研究,2012,16(16)：2939-2942.
[99]Hull TD, Ryu MH, Sullivan MJ, et al. Cyclic Di-GMP phosphodiesterases RmdA and RmdB are involved in regulating colony morphology and development in Streptomyces coelicolor [J]. J Bacteriol,2012,194(17):4642-4651.
[100]Chen W, Kuolee R, Yan H. The potential of 3',5'-cyclic diguanylic acid (c-di-GMP) as an effective vaccine adjuvant [J]. Vaccine,2010, 28(18):3080-3085.
[101]Podbielski A, Spellerberg B, Woischnik M, et al. Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci(GAS) [J]. Gene,1996,177:137-147.
[102]Li Z, Chen JH, Hao Y, et al. Structures of the PelD cyclic diguanylate effector involved in pellicle formation in Pseudomonas aeruginosa PAO1 [J]. J Biol Chem,2012,287(36):30191-30204.
[103]Mueller TF, Mas VR. Microarray applications in nephrology with special focus on transplantation [J]. J Nephrol,2012,25(5):589-602.
[104]Fujita K, Fukuda M, Iwahashi H. Significance of comprehensive gene expression analysis for evaluation of biological effects of manufactured nanomaterials [J]. Nihon Eiseiqaku Zasshi,2012,67(3):390-395.
[105]Miller MB, Tang YW. Basic concepts of microarrays and potential applications in clinical microbiology [J]. Clin Microbiol Rev,2009,22(4):611-633.
[106]Procop GW. Molecular diagnostics for the detection and characterization of microbial pathogens [J]. Clin Infect Dis,2007,45(2):S99-S111.
[107]Demirci H, Gregory ST, Dahlberg AE, et al. Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA [J]. EMBO J,2007, 26(2):567-577.
[108]Anderson ES, Paulley JT, Gaines JM, et al. The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice. [J]. Infect Immun,2009,77(8):3466-3474.
[109]Kaluarachchi H, Siebel JF, Kaluarachchi-Duffy S, et al. Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD [J]. Biochemistry,2011, 50(49):10666-10677.
[110]Anjem A, Varghese S, Imlay JA. Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli [J]. Mol Microbiol, 2009,72(4):844-858.
[111]Mandal D, Woolf TB, Rao R. Manganese selectivity of pmrl, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6 Residue Asp778 is essential for cation transport [J]. J Biol Chem, 2000,275:23933-23938.
[112]Elizarov SM, Alekseeva MG, Novikov FN, et al. Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus [J]. Biochemistry,2012,77(11):1258-1265.
[113]Neumann S, Grosse K, Sourjik V, et al. Chemotactic signaling via carbohydrate phosphotransferase systems in Escherichia coli [J]. Proc Natl Acad Sci USA, 2012,109(30):12159-12164.
[114]Zeng L, Burne RA. Multiple sugar:phosphotransferase system permeases participate in catabolite modification of gene expression in Streptococcus mutans [J]. Mol Microbiol,2008,70(1):197-208.
[115]Webb AJ, Homer KA, Hosie AH. A phosphoenolpyruvate-dependent phosphotransferase system is the principal maltose transporter in Streptococcus mutans [J]. J Bacteriol,2007,189(8):3322-3327.
[116]Stairs CW, Roger AJ, Hampl V. Eukaryotic pyruvate formate lyase and its activating enzyme were acquired laterally from a Firmicute [J]. Mol Biol Evol, 2011,28(7):2087-2099.
[117]Ghosh AK, Jacobs-Lorena M. Surface-expressed enolases of Plasmodium and other pathogens [J]. Mem Inst Oswaldo Cruz,2011,106(1):85-90.
[118]Nakamura N, Dai Q, Williams J, et al. Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility [J]. Biol Reprod,2013,88(4):90.
[119]Klein MI, Xiao J, Lu B,et al. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics [J]. PloS One,2012,7(9):e45795.
[120]Fan X, Wang Z, Ji P, et al. RgpA DNA Vaccine Induces Antibody Response and Prevents Alveolar Bone Loss in Experimental Peri-implantitis [J]. J Periodontol, 2012,8(23):823.
[121]van de Sande FH, Azevedo MS, Lund RG, et al. An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies [J]. Biofouling,2011,27(9):1057-1063.
[122]Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases:role in extracellular matrix formation of cariogenic biofilms [J]. Caries Res,2011,45(1):69-86.
[123]Zijnge V, van Leeuwen MB, Degener JE, et al. Oral biofilm architecture on natural teeth [J]. PloS One,2010,5(2):e9321.
[124]Flemming HC, Wingender J. The biofilm matrix [J]. Nat Rev Microbiol,2010, 8(9):623-633.
[125]Kuramitsu HK. Virulence factors of mutans streptococci:role of molecular genetics [J]. Crit Rev Oral Biol Med,1993,4(2):159-176.
[126]Banas JA, Vickerman MM. Vickerman. Glucan-Binding Proteins of the Oral Streptococci [J]. Criti Rev Oral Biol Med,2003,14(2):89-99.
[127]Coulon C, Vinogradov E, Filloux A, et al. Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain Pseudomonas aeruginosa PA14[J]. PloS One,2010,5(12):e14220.
[128]Banas JA, Miller JD, Fuschino ME, et al. Evidence that accumulation of mutans in a biofilm reflects natural selection rather than stress-induced adaptive mutation [J]. Appl Environ Microbiol,2007,73(1):357-361.
[129]Ccahuana-Vasquez RA, Cury JA. S. mutans biofilm model to evaluate antimicrobial substances and enamel demineralization [J]. Braz Oral Res,2010, 24(2):135-141.
[2]Ko J, Ryu KS, Kim H, et al. Structure of PP4397 reveals the molecular basis for different c-di-GMP binding modes by Pilz domain proteins [J]. J Mol Biol,2010, 398(1):97-110.
[3]Kozlova EV, Khajanchi BK, Sha J, et al. Quorum sensing and c-di-GMP-dependent alterations in gene transcripts and virulence-associated phenotypes in a clinical isolate of Aeromonas hydrophila [J]. Microb Pathog, 2011,50(5):213-223.
[4]Amikam D, Galperin MY. PilZ domain is part of the bacterial c-di-GMP binding protein [J]. Bioinformatics,2006,22(1):3-6.
[5]Lory S, Merighi M, Hyodo M. Multiple activities of c-di-GMP in Pseudomonas aeruginosa [J]. Nucleic Acids Symp Ser,2009,53(1):51-52.
[6]Newell PD, Monds RD, O'Toole GA. LapD is a bis-(3',5')-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1 [J]. Proc Natl Acad Sci USA,2009,106(9):3461-3466.
[7]Ueda A, Wood TK. Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885) [J]. PLoS Pathog,2009,5(6):e1000483.
[8]Karaolis DK, Rashid MH, Chythanya R, et al. c-di-GMP (3'-5'-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation [J]. Antimicrob Agents Chemother,2005,49(3):1029-1038.
[9]Frederick JR, Sarkar J, McDowell JV, et al. Molecular signaling mechanisms of the periopathogen, Treponema denticola [J]. J Dent Res,2011,90(10):1155-1163.
[10]Schirmer T, Jenal U. Structural and mechanistic determinants of c-di-GMP signalling [J]. Nat Rev Microbiol,2009,7(10):724-735.
[11]Yan W, Qu T, Zhao H, et al. The effect of c-di-GMP (3'-5'-cyclic diguanylic acid) on the biofilm formation and adherence of Streptococcus mutans [J]. Microbiol Res,2010,165(2):87-96.
[12]闫文娟,吴补领,屈铁军,等.外源性c-di-GMP对变形链球菌产酸、耐酸能力的影响[J].牙体牙髓牙周病学杂志,2009,19(9)：510-512.
[13]Dong C, Zhang FQ. Effect of denture base materials on mRNA expression of the adhesion-associated genes from the Streptococcus mutans biofilms [J]. J Oral Rehabil,2009,36(12):894-901.
[14]Wen ZT, Burne RA. LuxS-mediated signaling in Streptococcus mutans is involved in regulation of acid and oxidative stress tolerance and biofilm formation [J]. J Bacteriol,2004,186(9):2682-2691.
[15]Wen ZT, Nguyen AH, Bitoun JP, et al. Transcriptome analysis of LuxS-deficient Streptococcus mutans grown in biofilms [J]. Mol Oral Microbiol,2011, 26(1):2-18.
[16]Shemesh M, Tam A, Kott-Gutkowski M, et al. DNA-microarrays identification of Streptococcus mutans genes associated with biofilm thickness [J]. BMC Microbiol,2008,8:236.
[17]Nguyen T, Zhang Z, Huang IH, et al. Genes involved in the repression of mutacin I production in Streptococcus mutans [J]. Microbiology,2009,155(2):551-556.
[18]闫文娟,徐树军,谢苗苗,等.外源性单磷酸乌苷环二聚体对变形链球菌基因表达的影响[J].中国组织工程研究,2012,16(8)：1451-1454.
[19]Galperin MY, Nikolskaya AN, Koonin EV. Novel domains of the prokaryotic two-component signal transduction systems [J]. FEMS Microbiol Lett,2001, 203:11-21.
[20]Ross P, Weinhouse H, Aloni Y, et al. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid [J]. Nature,1987,325(6101): 279-281.
[21]Amikam D, Benziman M. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens [J]. J Bbacteriol,1989,171(12):6649-6655.
[22]Srivastava D, Waters CM. A tangled web:regulatory connections between quorum sensing and cyclic Di-GMP [J]. Journal of bacteriology,2012,194(17): 4485-4493.
[23]Sintim HO, Smith JA, Wang J, et al. Paradigm shift in discovering next-generation anti-infective agents_ targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules [J]. Future Med Chem,2010,2(6):1005-1035.
[24]Shanahan CA, Strobel SA. The bacterial second messenger c-di-GMP:probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs [J]. Org Biomol Chem,2012,10(46):9113-9129.
[25]Tchigvintsev A, Xu X, Singer A, et al. Structural insight into the mechanism of cyclic di-GMP hydrolysis by EAL domain phosphodiesterases [J]. J Mol Biol, 2010,402(24):524-538.
[26]Jenal U. Cyclic di-guanosine-monophosphate comes of age:a novel secondary messenger involved in modulating cell surface structures in bacteria? [J]. Curr Opin Microbiol,2004,7:185-191.
[27]Boyd CD, O'Toole GA. Second messenger regulation of biofilm formation: breakthroughs in understanding c-di-GMP effector systems [J]. Annu Rev Cell Dev Biol,2012,28:439-462.
[28]Rao F, Pasunooti S, Ng Y, et al. Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase [J]. Anal Biochem,2009,389(15):138-142.
[29]Navarro MV, De N, Bae N, et al. Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX [J]. Structure,2009, 17(8):1104-1116.
[30]Bordeleau E, Fortier LC, Malouin F, et al. c-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases [J]. PLoS Genet,2011,7(3):e1002039.
[31]Romling U, Gomelsky M, Galperin MY. C-di-GMP:the dawning of a novel bacterial signalling system [J]. Mol Microbiol,2005,57(3):629-639.
[32]Sondermann H, Shikuma NJ, Yildiz FH. You've come a long way:c-di-GMP signaling [J]. Current opinion in microbiology,2012,15(2):140-146.
[33]Jonas K, Melefors O, Romling U. Regulation of c-di-GMP metabolism in biofilms [J]. Future Microbiol,2009,4(3):341-358.
[34]Ryan RP, Dow JM. Intermolecular interactions between HD-GYP and GGDEF domain proteins mediate virulence-related signal transduction in Xanthomonas campestris [J]. Virulence,2010, 1(5):404-408.
[35]Galperin MY. A census of membrane-bound and intracellular signal transduction proteins in bacteria:bacterial IQ, extroverts and introverts [J]. BMC Microbiol, 2005,5:35.
[36]Yildiz FH, Visick KL. Vibrio biofilms:so much the same yet so different [J]. Trends Microbiol,2009,17(3):109-118.
[37]Nakhamchik A, Wilde C, Rowe-Magnus DA. Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus [J]. Appl Environ Microbiol,2008, 74(13):4199-4209.
[38]Krasteva PV, Fong JC, Shikuma NJ, et al. Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP [J]. Science,2010, 327(5967):866-868.
[39]Sun S, Kjelleberg S, McDougald D. Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists [J]. PloS One,2013,8(2):e56338.
[40]Tischler AD, Camilli A. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation [J]. Mol Microbiol,2004,53(3):857-869.
[41]Pratt JT, McDonough E, Camilli A. PhoB regulates motility, biofilms, and cyclic di-GMP in Vibrio cholerae [J]. J Bacteriol,2009,191(21):6632-6642.
[42]Li TN, Chin KH, Fung KM, et al. A Novel Tetrameric PilZ Domain Structure from Xanthomonads [J]. PloS One,2011,6(7):e22036.
[43]Whitney JC, Colvin KM, Marmont LS, et al. Structure of the cytoplasmic region of PelD, a degenerate diguanylate cyclase receptor that regulates exopolysaccharide production in Pseudomonas aeruginosa [J]. J Biol Chem,2012, 287(28):23582-23593.
[44]Jimenez PN, Koch G, Thompson JA, et al. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa [J]. Microbiol Mol Biol Rev, 2012,76(1):46-65.
[45]Lee VT, Matewish JM, Kessler JL, et al. A cyclic-di-GMP receptor required for bacterial exopolysaccharide production [J]. Mol Microbiol,2007, 65(6):1474-1484.
[46]Wolfe AJ, Visick KL. Get the message out:cyclic-Di-GMP regulates multiple levels of flagellum-based motility [J]. J Bacteriol,2008,190(2):463-475.
[47]Hay ID, Remminghorst U, Rehm BH. MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa [J]. Appl Environ Microbiol,2009,75(4):1110-1120.
[48]Jain R, Behrens AJ, Kaever V, et al. Type IV pilus assembly in Pseudomonas aeruginosa over a broad range of cyclic di-GMP concentrations [J]. J Bacteriol, 2012,194(16):4285-4294.
[49]Coggan KA, Wolfgang MC. Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype [J]. Curr Issues Mol Biol,2012,14(2):47-70.
[50]Guo Y, Rowe-Magnus DA. Identification of a c-di-GMP-regulated polysaccharide locus governing stress resistance and biofilm and rugose colony formation in Vibrio vulnificus [J]. Infect Immun,2010,78(3):1390-1402.
[51]Abel S, Chien P, Wassmann P, et al. Regulatory cohesion of cell cycle and cell differentiation through interlinked phosphorylation and second messenger networks [J]. Mol Cell,2011,43(4):550-560.
[52]Sultan SZ, Pitzer JE, Boquoi T, et al. Analysis of the HD-GYP domain cyclic dimeric GMP phosphodiesterase reveals a role in motility and the enzootic life cycle of Borrelia burgdorferi [J]. Infect Immun,2011,79(8):3273-3283.
[53]Chen MW, Kotaka M, Vonrhein C, et al. Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling [J]. J Bacteriol,2012,194(18):4837-4846.
[54]Roy AB, Petrova OE, Sauer K. The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion [J]. J Bacteriol,2012, 194(11):2904-2915.
[55]Sun YC, Koumoutsi A, Jarrett C, et al. Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases [J]. PloS One,2011,6(4):e19267.
[56]Kirillina O, Fetherston JD, Bobrov AG, et al. HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis [J]. Mol Microbiol,2004,54:75-88.
[57]Hickman JW, Tifrea DF, Harwood CS. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels [J]. Proc Natl Acad Sci USA,2005,102:14422-14427.
[58]Purcell EB, McKee RW, McBride SM, et al. Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile [J]. J Bacteriol,2012, 194(13):3307-3316.
[59]Wang KC, Hsu YH, Huang YN, et al. A previously uncharacterized gene stm0551 plays a repressive role in the regulation of type 1 fimbriae in Salmonella enterica serotype Typhimurium [J]. BMC microbiol,2012,12:111.
[60]Wang H, Wu JH, Ayala JC, et al. Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease [J]. J Bacteriol,2011,193(23):6529-6538.
[61]Mikami Y, Suzuki N, Takahashi T, et al. Bacitracin Upregulates mbrAB Transcription via mbrCD to Confer Bacitracin Resistance in Streptococcus mutans [J]. J Pharmacol Sci,2011,117(3):204-207.
[62]Beyhan S, Tischler AD, Camilli A, et al. Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di-GMP level [J]. J Bacteriol,2006, 188(10):3600-3613.
[63]Kajfasz JK, Martinez AR, Rivera-Ramos I, et al. Role of Clp proteins in expression of virulence properties of Streptococcus mutans [J]. J Bacteriol,2009, 191(7):2060-2068.
[64]Trimble MJ, McCarter LL. Bis-(3'-5')-cyclic dimeric GMP-linked quorum sensing controls swarming in Vibrio parahaemolyticus [J]. Proc Natl Acad Sci USA,2011,108(44):18079-18084.
[65]Chong P, Chattoraj P, Biswsa I. Activation of the SMU.1882 Transcription by CovR in Streptococcus mutans [J]. PloS One,2012,5(11):e15528.
[66]Shemesh M, Tam A, Aharoni R, et al. Genetic adaptation of Streptococcus mutans during biofilm formation on different types of surfaces [J]. BMC Microbiol,2010,10:51.
[67]Lim B, Beyhan S, Yildiz FH. Regulation of Vibrio polysaccharide synthesis and virulence factor production by CdgC, a GGDEF-EAL domain protein, in Vibrio cholerae [J]. J Bacteriol,2007,189(3):717-729.
[68][68] Shikuma NJ, Fong JC, Yildiz FH. Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT [J]. PLoS pathogens,2012,8(5):e 1002719.
[69]Ryan RP, McCarthy Y, Andrade M, et al. Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris [J]. Proc Natl Acad Sci USA,2010, 107(13):5989-5994.
[70]Ahmad I, Lamprokostopoulou A, Le Guyon S, et al. Complex c-di-GMP Signaling Networks Mediate Transition between Virulence Properties and Biofilm Formation in Salmonella enterica Serovar Typhimurium [J]. PloS One, 2011,6(12):e28351.
[71]Hisert KB, MacCoss M, Shiloh MU, et al. A glutamatealanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages:role of cyclic diGMP [J]. Mol Microbiol, 2005,56(5):1234-45.
[72]Chung IY, Choi KB, Heo YJ, et al. Effect of PEL exopolysaccharide on the wspF mutant phenotypes in Pseudomonas aeruginosa PA14 [J]. J Microbiol Biotechnol, 2008,18(7):1227-1234.
[73]Kulasakara H, Lee V, Brencic A, et al. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence [J]. Proc Natl Acad Sci USA,2006, 103(8):2839-44.
[74]Deng Y, Schmid N, Wang C, et al. Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover [J]. Proc Natl Acad Sci USA,2012, 109(38):15479-15484.
[75]Petters T, Zhang X, Nesper J, et al. The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus [J]. Mol Microbiol,2012,84(1):147-165.
[76]Romling U, Amikam D. C-di-GMP is a ubiquitous second messenger that controls the transition from a free-living, motile lifestyle to a biofilm lifestyle in many bacteria [J]. Curr Opin Microbiol,2006,9(2):218-228.
[77]Navarro MV, Newell PD, Krasteva PV, et al. Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis [J]. PLoS Biol,2011,9(2):e1000588.
[78]Yang CY, Chin KH, Chuah ML, et al. The structure and inhibition of a GGDEF diguanylate cyclase complexed with (c-di-GMP)(2) at the active site [J]. Acta Crystallogr D Biol Crystallogr,2011,67(12):997-1008.
[79]Benach J, Swaminathan SS, Tamayo R, et al. The structural basis of cyclic diguanylate signal transduction by PilZ domains [J]. EMBO J,2007, 26(24):5153-5166.
[80]Fang X, Gomelsky M. A post-translational, c-di-GMP-dependent mechanism regulating flagellar motility [J]. Mol Microbiol,2010,76:1295-1305.
[81]Srivastava D, Harris RC, Waters CM. Integration of cyclic di-GMP and quorum sensing in the control of vpsT and aphA in Vibrio cholerae [J]. J Bacteriol,2011, 193(22):6331-6341.
[82]Rehman ZU, Rehm BH. Dual Roles of Pseudomonas aeruginosa AlgE in Secretion of the Virulence Factor Alginate and Formation of the Secretion Complex [J]. Appl Environ Microbiol,2013,79(6):2002-2011.
[83]Leduc JL, Roberts GP. Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri. [J]. J Bacteriol,2009, 191:7121-7122.
[84]Baird NJ, Kulshina N, Ferre-D'Amare AR. Riboswitch function:flipping the switch or tuning the dimmer? [J]. RNA Biol,2010,7(3):328-332.
[85]Beyhan S, Odelland LS, Yildiz FH. Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae [J]. J Bacteriol,2008,190:7392-7405.
[86]Liberman JA, Wedekind JE. Base ionization and ligand binding:how small ribozymes and riboswitches gain a foothold in a protein world [J]. Curr Opin Struct Biol,2011,21 (3):327-334.
[87]Petrova OE, Sauer K. Dispersion by Pseudomonas aeruginosa requires an unusual posttranslational modification of Bd1A [J]. Proc Natl Acad Sci USA, 2012,109(41):16690-16695.
[88]Massiea JP, Reynoldsa EL, Koestlera BJ, et al. Quantification of high-specificity cyclic diguanylate signaling [J]. Proc Natl Acad Sci USA,2012, 109(31):12746-12751.
[89]Kim JS, Shin DH. Inhibitory effect on Streptococcus mutans and mechanical properties of the chitosan containing composite resin [J]. Restor Dent Endod, 2013,38(1):36-42.
[90]Mohamed Hamouda I. Current perspectives of nanoparticles in medical and dental biomaterials [J]. J Biomed Res,2012,26(3):143-151.
[91]闫文娟,杨德鸿,吴补领.外源性单磷酸鸟苷环二聚体防龋的动物实验研究[J].南方医科大学学报,2012,32(5)：639-642.
[92]闫文娟.c-di-GMP信号通路在变形链球菌致龋过程中作用的初步研究[J]. 第四军医大学,2009：73-76.
[93]Ajdic D, McShan WM, McLaughlin RE, et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen [J]. Proc Natl Acad Sci USA,2002,99(22):14434-14439.
[94]Zhao L, KuoLee R, Harris G, et al. c-di-GMP protects against intranasal Acinetobacter baumannii infection in mice by chemokine induction and enhanced neutrophil recruitment [J]. Int Immunopharmacol,2011, 11(9):1378-1383.
[95]Koyanagi S, LevesqueCM. Characterization of a Streptococcus mutans Intergenic Region Containing a Small Toxic Peptide and Its cis-Encoded Antisense Small RNA Antitoxin [J]. PloS One,2012,8(1):e54291.
[96]Madhun AS, Haaheim LR, Nostbakken JK, et al. Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Thl CD4+ cells and strong mucosal and systemic antibody responses in mice [J]. Vaccine,2011,29(31):4973-4982.
[97]Costa MT, Dorta ML, Ribeiro-Dias F, et al. Biofilms of black tooth stains:PCR analysis reveals presence of Streptococcus mutans [J]. Braz Dent J,2012, 23(5):555-558.
[98]闫文娟.外源性单磷酸鸟苷环二聚体抑制变形链球菌生物膜的形成能力[J].中国组织工程研究,2012,16(16)：2939-2942.
[99]Hull TD, Ryu MH, Sullivan MJ, et al. Cyclic Di-GMP phosphodiesterases RmdA and RmdB are involved in regulating colony morphology and development in Streptomyces coelicolor [J]. J Bacteriol,2012,194(17):4642-4651.
[100]Chen W, Kuolee R, Yan H. The potential of 3',5'-cyclic diguanylic acid (c-di-GMP) as an effective vaccine adjuvant [J]. Vaccine,2010, 28(18):3080-3085.
[101]Podbielski A, Spellerberg B, Woischnik M, et al. Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci(GAS) [J]. Gene,1996,177:137-147.
[102]Li Z, Chen JH, Hao Y, et al. Structures of the PelD cyclic diguanylate effector involved in pellicle formation in Pseudomonas aeruginosa PAO1 [J]. J Biol Chem,2012,287(36):30191-30204.
[103]Mueller TF, Mas VR. Microarray applications in nephrology with special focus on transplantation [J]. J Nephrol,2012,25(5):589-602.
[104]Fujita K, Fukuda M, Iwahashi H. Significance of comprehensive gene expression analysis for evaluation of biological effects of manufactured nanomaterials [J]. Nihon Eiseiqaku Zasshi,2012,67(3):390-395.
[105]Miller MB, Tang YW. Basic concepts of microarrays and potential applications in clinical microbiology [J]. Clin Microbiol Rev,2009,22(4):611-633.
[106]Procop GW. Molecular diagnostics for the detection and characterization of microbial pathogens [J]. Clin Infect Dis,2007,45(2):S99-S111.
[107]Demirci H, Gregory ST, Dahlberg AE, et al. Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA [J]. EMBO J,2007, 26(2):567-577.
[108]Anderson ES, Paulley JT, Gaines JM, et al. The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice. [J]. Infect Immun,2009,77(8):3466-3474.
[109]Kaluarachchi H, Siebel JF, Kaluarachchi-Duffy S, et al. Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD [J]. Biochemistry,2011, 50(49):10666-10677.
[110]Anjem A, Varghese S, Imlay JA. Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli [J]. Mol Microbiol, 2009,72(4):844-858.
[111]Mandal D, Woolf TB, Rao R. Manganese selectivity of pmrl, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6 Residue Asp778 is essential for cation transport [J]. J Biol Chem, 2000,275:23933-23938.
[112]Elizarov SM, Alekseeva MG, Novikov FN, et al. Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus [J]. Biochemistry,2012,77(11):1258-1265.
[113]Neumann S, Grosse K, Sourjik V, et al. Chemotactic signaling via carbohydrate phosphotransferase systems in Escherichia coli [J]. Proc Natl Acad Sci USA, 2012,109(30):12159-12164.
[114]Zeng L, Burne RA. Multiple sugar:phosphotransferase system permeases participate in catabolite modification of gene expression in Streptococcus mutans [J]. Mol Microbiol,2008,70(1):197-208.
[115]Webb AJ, Homer KA, Hosie AH. A phosphoenolpyruvate-dependent phosphotransferase system is the principal maltose transporter in Streptococcus mutans [J]. J Bacteriol,2007,189(8):3322-3327.
[116]Stairs CW, Roger AJ, Hampl V. Eukaryotic pyruvate formate lyase and its activating enzyme were acquired laterally from a Firmicute [J]. Mol Biol Evol, 2011,28(7):2087-2099.
[117]Ghosh AK, Jacobs-Lorena M. Surface-expressed enolases of Plasmodium and other pathogens [J]. Mem Inst Oswaldo Cruz,2011,106(1):85-90.
[118]Nakamura N, Dai Q, Williams J, et al. Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility [J]. Biol Reprod,2013,88(4):90.
[119]Klein MI, Xiao J, Lu B,et al. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics [J]. PloS One,2012,7(9):e45795.
[120]Fan X, Wang Z, Ji P, et al. RgpA DNA Vaccine Induces Antibody Response and Prevents Alveolar Bone Loss in Experimental Peri-implantitis [J]. J Periodontol, 2012,8(23):823.
[121]van de Sande FH, Azevedo MS, Lund RG, et al. An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies [J]. Biofouling,2011,27(9):1057-1063.
[122]Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases:role in extracellular matrix formation of cariogenic biofilms [J]. Caries Res,2011,45(1):69-86.
[123]Zijnge V, van Leeuwen MB, Degener JE, et al. Oral biofilm architecture on natural teeth [J]. PloS One,2010,5(2):e9321.
[124]Flemming HC, Wingender J. The biofilm matrix [J]. Nat Rev Microbiol,2010, 8(9):623-633.
[125]Kuramitsu HK. Virulence factors of mutans streptococci:role of molecular genetics [J]. Crit Rev Oral Biol Med,1993,4(2):159-176.
[126]Banas JA, Vickerman MM. Vickerman. Glucan-Binding Proteins of the Oral Streptococci [J]. Criti Rev Oral Biol Med,2003,14(2):89-99.
[127]Coulon C, Vinogradov E, Filloux A, et al. Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain Pseudomonas aeruginosa PA14[J]. PloS One,2010,5(12):e14220.
[128]Banas JA, Miller JD, Fuschino ME, et al. Evidence that accumulation of mutans in a biofilm reflects natural selection rather than stress-induced adaptive mutation [J]. Appl Environ Microbiol,2007,73(1):357-361.
[129]Ccahuana-Vasquez RA, Cury JA. S. mutans biofilm model to evaluate antimicrobial substances and enamel demineralization [J]. Braz Oral Res,2010, 24(2):135-141.